Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes serious sexually transmitted diseases in humans. The study of chlamydial biology and pathogenesis is particularly challenging because of the lack of a system for inserting or inactivating target genes. In the absence of a genetic system, genome sequence analysis provides uniquely valuable information on the chlamydiae. Genomic approaches in this system are particularly feasible because sequencing the small, A + T rich, non-repetitive chlamydial genome can be completed at a comparatively small cost. This proposal describes the use of genome sequencing to investigate three novel chlamydial phenotypes that were identified following laboratory and epidemiologic analysis of isolates from the University of Washington Chlamydia Repository (UWCR). The UWCR contains over 12,000 clinical isolates and is maintained by the Laboratory Core of the STD-CRC. The phenotypes to be examined are the following. First, isolates within certain serovars are highly associated with rectal infection in homosexual men and, thus, demonstrate rectal tropism. Second, certain C. trachomatis isolates, particularly those of serovar G, form large numbers of secondary inclusions within cells infected with single elementary bodies. Finally, a group of C. trachomatis isolates has been identified that form non-fusogenic inclusions in tissue culture. The genetic basis for these phenotypes will be explored through the completion of the genome sequence for each of six representative clinical isolates. This sequencing will allow identification of nucleotide polymorphisms (NPs)among the isolates and in comparison to published chlamydial genomes. These NPs will then be ranked and a larger set of strains will be screened to statistically connect individual NPs with the phenotypes. These quantitative studies will be conducted in association with the STI-TM-CRC Biostatistics Core and will utilize recent clinical isolates selected from the UWCR. Tissue culture models and other in vitro approaches will then be used to examine the affected gene products as possible contributors to the phenotype. We anticipate that these studies will provide evidence linking chlamydial genotypes with the three phenotypic distinctions and will identify chlamydial genes important in previously uncharacterized aspects of chlamydial biology and pathogenesis. Completion of these genome sequences will be also important to tools for the study of chlamydial evolution and diversity.